Differential Adsorption and Inhibition of Cellulase Components Regulated by Hydrothermal Pretreatment-Induced Surface Lignins

Abstract

Nonproductive adsorption of cellulases onto lignin severely limits the enzymatic hydrolysis efficiency of lignocellulosic biomass. While differential adsorption across cellulase components is known, the spatial and conformational mechanisms by which pretreated lignin selectively deactivates specific enzymes remain poorly understood. Here, surface lignins isolated from hydrothermally pretreated poplar at different temperatures (HP170-SL and HP190-SL) were investigated to establish the molecular relationship between lignin structural remodeling and component-selective cellulase inhibition. Structural characterization showed that elevated pretreatment temperature induced extensive β-O-4 bond cleavage, molecular weight reduction, and surface charge intensification. Surface plasmon resonance analysis revealed that HP190-SL exhibited stronger adsorption affinity and binding stability toward cellobiohydrolase I (CBHI), whereas its interaction with endoglucanase (EG) was significantly weaker compared with HP170-SL. Circular dichroism and enzyme activity assays further demonstrated that HP190-SL induced more substantial conformational disruption and activity loss of CBHI than HP170-SL, while EG remained relatively stable under different lignin treatments. Molecular dynamics simulations suggested that HP190-SL formed a broader and more stable adsorption interface with CBHI through combined van der Waals and electrostatic interactions. These results demonstrate the molecular mechanism by which hydrothermal lignin restructuring selectively induces CBHI denaturation via active-site shielding and conformational perturbation. This work provides mechanistic insights into lignin-associated cellulase inhibition and may guide the development of improved biomass pretreatment and saccharification strategies.